The effects of ligands on enzymic carboxyl-methylation of neurophysins

Vol. 73, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

THE EFFECTS OF LIGANDS ON ENZYMIC CARBOXYL-METHYLATION Emanuel

J. Diliberto,

, Julius

Axelrod

and Irwin

M. Chaiken

Laboratory of Clinical Science National Institute of Mental Health and Laboratory of Chemical Biology Institute of Arthritis, Metabolism, and Digestive National Institutes of Health Bethesda, Maryland 20014

National

Received

*

Jr.

OF NEUROPHYSINS

October

Diseases

19,1976

Summary: The methyl-acceptor activities of bovine neurophysins I and II for the enzyme protein carboxymethylase (EC 2.1.1.24) were found to be similar and as high as for other previously identified, biologically active protein substrates. Effects on the rate of methylation of these neurophysins were investigated with the posterior pituitary hormone ligands, oxytocin and vasopressin, and the hormone-related tripeptide ligand, methionyl-tyrosylphenylalaninamide. An increase in the rate of neurophysin II methylation was observed with both oxytocin and tripeptide. This ligand-induced response did not occur with either native neurophysin I or disulfide-scrambled neurophysin :1. INTRODUCTION Protein

carboxymethylase

0-methyltransferase, the methyl formation bovine

group

EC 2.1.1.24;

methyl

gland

of the enzyme showed acceptors

while

not

as substrates

serve

active

proteins

esters

that

all

the posterior

showed

(3). that

protein

pituitary

the posterior

of neurophysin

carboxymethylase

temporal

chains

guinea

1063

pig

effective

methyl

and vasopressin,

for

did

biologically proteins,

protein

has been of endogenous hypothalamus.

National

from specificity

hormone-binding

substrates

of

in the

purified

were

of other

relationship

Present address: Laboratory of Neurochemistry, Mental Health, Bethesda, Md. 20014. G 1976 by Academic Press, Inc. of reproduction in any form reserved.

oxytocin

and the synthesis

in cultured

transfer

of the substrate

pituitary

effective

*

Copyright All rights

side

hormones

investigation

a close

for

A study

However,

Further,

catalyzes

The enzyme has been

hormones,

methylase

the synthesis

(l-4).

pituitary

as highly

II)

to carboxyl

anterior

do serve

between

methylase

and characterized.

the neurophysins, (5,6).

protein

of S-adenosyl-L-methionine

of protein pituitary

(S-adenosyl-L-methionine:protein-carboxyl

Institute

carboxy-

found

(7)

substrate This of

Vol. 73, No. 4, 1976

relationship

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is perhaps an expression of -in vivo methylation

of neurophysin

by this enzyme. Inasmuch as neurophysins are active and neurophysin hormone-binding activity

substrates for protein carboxymethylase appears to involve

an ionic inter-

action between a side-chain carboxyl group of neurophysin and the a-amino group of hormone or other peptide ligand (8), a role for methylation neurophysin function

must be considered.

by tht? high specific

activity

One aspect requiring

clarification

methylation

[lysine-vasopressinl-Sepharose

is suggested further

of the enzyme in the posterior

and hormone binding.

of neurophysin did not affect

This possibility

is the direct Previously

qualitatively

in

relationship,

pituitary

(4).

if any, between

(6), it was shown that methylation the binding of the protein

by all-or-none

affinity

to

chromatographic elution.

In the present study, the reverse question was raised, namely, does ligand binding affect related

methylation.

tripeptide

We find that binding of oxytocin

increase carboxyl-methylation

and a hormone-

of neurophysin II.

MATERIALSANDMETHODS Enzyme Purification and Assay-- Protein carboxymethylase was purified from bovine pituitaries by the procedure previously described (3,4). Protein carboxymethylase activity assays (4) were carried out at pH 6.0. In most experiments, neurophysins were preinsubated with ligand at 20" for 10 min prior to addition of S-adenosyl-L-[methylHlmethionine and purified protein carboxymethylase (more than 2000-fold purified); this enzyme incubation was carried out at 37' for 15 min (the period of linear activity). Specific enzqic activity is expressed as units per mg of protein, where a unit equals 1 pmol [ HImethyl transferred per 15 min. Neurophysins-- Bovine neurophysins I and II were isolated from acetonedried posterior pituitaries (Pel-Freeze, Rogers, Arkansas) by the procedure of Hollenberg and Hope (9) with final purification using DEAE-SephadexA-50 (10). Disulfide-scrambled neurophysin II was prepared by incubation of 0.6 mg of neurophysin II in 1 ml of 0.1 M ammonium bicarbonate, pH 7.2, containing 1.0 mM 2-mercaptoethanol for 45 h at room temperature. The denatured product was used after exhaustive lyophilization. Synthetic oxytocin (generously Neurophysin Ligands and Other Materialsdesalted provided by Sandoz Pharmaceuticals, as "Syntocinon") was lyophilized, by passing through a Sephadex G-15 column equilibrated with 50% acetic acid, and lyophilized repeatedly from water before use. Native arginine-vasopressin was isolated as a byproduct of the neurophysin isolation and purified further on sulfoethyl-Sephadex C-25 with an elution procedure similar to that described previously (11). Methionyl-tyrosyl-phenylalaninamide, a tripeptide, was prepared by the solid phas3 synthesis method (12). S-adenosyl-L-[methylHlmethionine, 8.54 Ci/mmole, was purchased from New England Nuclear Corp. Luteinizing hormone (ovine) was a generous gift of Dr. Leo Reichert, Emory University.

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BIOCHEMICAL

Vol. 73, No. 4,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

1

Comparison of the effects of oxytocin and methionyl-tyrosylphenylalaninamide on the carboxyl-methylation of neurophysins. Protein

PM Activity Ratio with/without added ligand

PCM Activityb (with no additions) (units)

Substrate

a

Oxytocin (0.6 mM) Neurophysin II 10 pM 20 UM

l.62d 3.41d

1.68 1.14

1.74 1.14

Neurophysin 11 (Scrambled) 10 IJM 20 nM

0.53e 1.33e

-------

1.08 0.85

Neurophysin I 10 I-IM 20 UM

2.24 4.04

0.75 0.86

0.83 0.99

Luteining hormone 10 nM

2.29

1.07

----

aUnless otherwise of results from b

Protein Materials

indicated, data are from one experiment at least two other experiments.

carboxymethylase and Methods.

(PCM) activity

was assayed

and are

as described

typical in

%ethionyl-tyrosyl-phenylalaninamide. d

Average

of three

eThese data neurophysin respectively.

experiments.

were obtained II activities

in an experiment where the control native were 1.28 and 2.58 units for 10 uM and 20 !JM,

RESULTS Effect pituitary

of ligand hormones,

carboxymethylase of bovine The relative trations

binding oxytocin

(3).

neurophysin increase of neurophysin

on methylation and vasopressin,

However, II

as shown

was found

in methylation II,

of neurophysins--

reflecting

are not

in Table

1065

rate

for

greater

a decrease

protein

of methylation

by the presence

was consistently at least

substrates

1, the

to be increased

The posterior

of oxytocin

at lower in Km.

concen-

Such a

Vol. 73, No. 4, 1976

response

BIOCHEMICAL

was not observed

hormone

(LH).

An indication

in the observation nificantly for

that

the rate

neurophysin

reduced

in

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

non-oxytocin of the specificity

the rate

the presence

of neurophysin

of methylation

of oxytocin

examined

for

its

an increase that

in a manner effect

found

with

assay

were

studies

Comparison

by this

were

response--

which

caused

an increase

II

or slightly

to bind

II

specifically

(13), (Table

to an extent

peptide

was 1).

Again,

similar

effect

(data

with

based

to

activity

on the errors

rate the

II

on substrate II

in

of the

decrease

activity

was shown previously

Denaturation

(13).

a marked

of neurophysin

in substrate

II

activity

of methionyl-tyrosyl-phenylalaninamide of methylation

scrambled

in altering not

in methylating

neurophysin

ligands

caused

in the

changes

neurophysin

a concentration

was ineffective

neurophysin

Also,

as insignificant.

and denatured

procedure

had no significant oxytocin

sig-

shown).

and vasopressin

to interpret

Disulfide-scrambled

Furthermore,

1).

shown

of neurophysin

effects,

regarded

in binding

reduction

(Table

not

changed

was obtained,

difficult

of native

to be inactive

to oxytocin

of ligand

considered

and therefore

and ligand

was obtained

oxytocin.

In the above 15% or less

effect in altering

(data

previously

of methylation

luteinizing

1).

on the methylation

in the rate

II

was little

(Table

similar

i.e.,

was ineffective

Methionyl-tyrosyl-phenylalaninamide, to neurophysins

proteins,

of the ligand

arginine-vasopressin

of methylation

I,

binding

of native

neurophysin

the rate

neurophysin

(Table

of methylation

1).

II

Similarly,

of scrambled

shown).

DISCUSSION The side-chain hormone

binding

methylase. physins, sites.

carboxyl (8)

is a potential

However, hormone This

view

group

since

binding is

of neurophysin site

the hormone

for

methylation

does not

and carboxyl-methylation

consistent

with

a previous

1066

purported

by protein

inhibit

methylation

probably report

to be involved

occur

in which

in

carboxyof neuroat different methylation

of

Vol. 73, No. 4, 1976

neurophysin

BIOCHEMICAL

did

not

appear

vasopressinl-Sepharose The data ligands

not

i.e.,

they

the present

study

inhibit

II

only,

physin,

allowing

for

Aspects

of conformation aggregation

The high

(13)

substrate

effect

protein

carboxymethylase

neurophysin

[lysine-

a greater could

activity

the idea

in fact

certain

neurophysin

have an opposite Furthermore,

effect, a degree

of a significant

and methionyl-tyrosyl-phenylalaninamide I.

This

phenomenon

in some conformational affinity

by ligand

a particular

feature

of neurophysins

(5),

and the high

in the posterior of a biological

pituitary role

aspect

of neurophysin

be affected

on carboxyl-methylation

with

to

by the observation

on neurophysin

or perhaps

that

of reaction.

indicated

oxytocin

change

which

but

the rate

is

and not

at least

hormone

consistent

in

with

of a hormone-induced

simply

of the protein

indicate

methylation

response

of methylation

on neurophysin

the binding

an increase

in this

stimulation

result

in

do not

produce

of specificity

to alter

RESEARCH COMMUNICATIONS

(6).

obtained only

AND BIOPHYSICAL

for

may be the of the neuro-

for

binding

the enzyme. might

in monomeric the specificity

specific

activity

and hypothalamus methylation

include

folding. of the of (4)

are

in hormone-

function.

REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13.

Kim, S. and Paik, W.K. (1971) Biochemistry, 10, 3141-3145. Morin, A.M. and Liss, M. (1973) Biochem. Biophys. Res. Commun., 52, 373-378. Diliberto, E.J., Jr. and Axelrod, J. (1974) Proc. Natn. Acad. Sci. U.S.A., 71, 1701-1704. Diliberto, E.J., Jr. and Axelrod, J. (1976) J. Neurochem., 26, 1159-1165. Axelrod, J. and Diliberto, E.J., Jr. (1975) Ann. N.Y. Acad. Sci., 248, 90-91. Edgar, D.H. and Hope, D.B. (1974) FEBS Letters, 49, 145-148. Kim, S., Pearson, D. and Paik, W.P. (1975) Biochem. Biophys. Res. Commun., 67, 448-454. Walter, R. and Hoffman, P.L. (1973) Fed. Proc., 32, 5674. Hollenberg, M.D. and Hope, D.B. (1968) Biochem. J., 106, 557-564. Breslow, E., Aanning, H.L., Abrash, L. and Schmir, M. (1971) ,J. Biol. Chem., 246, 5179-5188. Acher, R., Light, A. and duvigneaud, V. (1958) J. Biol. Chem., 233, 116-120. Merrifield, R.B. (1963) J. Amer. Chem. Sot., 85, 2149-2154. Chaiken, I.M., Randolph, R.E. and Taylor, H.C. (1975) Ann. N.Y. Acad. Sci., 248, 442-450.

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